Immunoregulatory mechanisms of macrophage PPAR-γ in mice with experimental inflammatory bowel disease.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is widely expressed in macrophages and has been identified as a putative target for the development of novel therapies against inflammatory bowel disease (IBD). Computational simulations identified macrophages as key targets for therapeutic interventions against IBD. This study aimed to characterize the mechanisms underlying the beneficial effects of macrophage PPAR-γ in IBD. Macrophage-specific PPAR-γ deletion significantly exacerbated clinical activity and colonic pathology, impaired the splenic and mesenteric lymph node regulatory T-cell compartment, increased percentages of lamina propria (LP) CD8+ T cells, increased surface expression of CD40, Ly6C, and Toll-like receptor 4 (TLR-4) in LP macrophages, and upregulated expression of colonic IFN-γ, CXCL9, CXCL10, IL-22, IL1RL1, CCR1, suppressor of cytokine signaling 3, and MHC class II in mice with IBD. Moreover, macrophage PPAR-γ was required for accelerating pioglitazone-mediated recovery from dextran sodium sulfate (DSS) colitis, providing a cellular target for the anti-inflammatory effects of PPAR-γ agonists in IBD.

Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses.

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.

An emerging cyberinfrastructure for biodefense pathogen and pathogen-host data.

The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host-pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/.

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case.

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Bioinformatics for rice resources.

The distinguishing feature of the 'new biology' is that it is information intensive. Not only does it demand access to and assimilation of vast data sets accumulated by engineered laboratory processes, but it also demands a previously unimaginable level of data integration across data types and sources. There are various information resources available for rice. In addition, there are various information resources that are not focused on rice but that contain rice data. The challenge for rice researchers and breeders is to access this wealth of data meaningfully. This challenge will grow significantly as international efforts aimed at sequencing the entire rice genome come into full swing. Only through concerted efforts in bioinformatics will the power of these public data be brought to bear on the needs of rice researchers and breeders worldwide. These efforts will need to focus on two large but distinct areas: (1) development of an effective bioinformatics infrastructure (hardware systems, software systems, and software engineers and support staff) and (2) computational biology research in visualization and analysis of very large, complex data sets, such as those that will be developed using high-throughput expression technologies, large-scale insertional mutagenesis, and biochemical profiling of various types. In the midst of the large flow of high-throughput data that the international rice genome sequencing efforts will produce, it is also imperative that integration of those data with unique germplasm data held in trust by the CGIAR be a part of the informatics infrastructure. This paper will focus on the state of rice information resources, the needs of the rice community, and some proposed bioinformatics activities to support these needs.

ISYS: a decentralized, component-based approach to the integration of heterogeneous bioinformatics resources.

MOTIVATION: Heterogeneity of databases and software resources continues to hamper the integration of biological information. Top-down solutions are not feasible for the full-scale problem of integration across biological species and data types. Bottom-up solutions so far have not integrated, in a maximally flexible way, dynamic and interactive graphical-user-interface components with data repositories and analysis tools. RESULTS: We present a component-based approach that relies on a generalized platform for component integration. The platform enables independently-developed components to synchronize their behavior and exchange services, without direct knowledge of one another. An interface-based data model allows the exchange of information with minimal component interdependency. From these interactions an integrated system results, which we call ISYSf1.gif" BORDER="0">. By allowing services to be discovered dynamically based on selected objects, ISYS encourages a kind of exploratory navigation that we believe to be well-suited for applications in genomic research.

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant.

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).

Comparative analysis of expressed sequences in Phytophthora sojae.

Phytophthora sojae (Kaufmann and Gerdemann) is an oomycete that causes stem and root rot on soybean (Glycine max L. Merr) plants. We have constructed three cDNA libraries using mRNA isolated from axenically grown mycelium and zoospores and from tissue isolated from plant hypocotyls 48 h after inoculation with zoospores. A total of 3,035 expressed sequence tags (ESTs) were generated from the three cDNA libraries, representing an estimated 2,189 cDNA transcripts. The ESTs were classified according to putative function based on similarity to known proteins, and were analyzed for redundancy within and among the three source libraries. Distinct expression patterns were observed for each library. By analysis of the percentage G+C content of the ESTs, we estimate that two-thirds of the ESTs from the infected plant library are derived from P. sojae cDNA transcripts. The ESTs originating from this study were also compared with a collection of Phytophthora infestans ESTs and with all other non-human ESTs to assess the similarity of the P. sojae sequences to existing EST data. This collection of cDNA libraries, ESTs, and accompanying annotation will provide a new resource for studies on oomycetes and on soybean responses to pathogen challenge.

The phytophthora genome initiative database: informatics and analysis for distributed pathogenomic research.

The Phytophthora Genome Initiative (PGI) is a distributed collaboration to study the genome and evolution of a particularly destructive group of plant pathogenic oomycete, with the goal of understanding the mechanisms of infection and resistance. NCGR provides informatics support for the collaboration as well as a centralized data repository. In the pilot phase of the project, several investigators prepared Phytophthora infestans and Phytophthora sojae EST and Phytophthora sojae BAC libraries and sent them to another laboratory for sequencing. Data from sequencing reactions were transferred to NCGR for analysis and curation. An analysis pipeline transforms raw data by performing simple analyses (i.e., vector removal and similarity searching) that are stored and can be retrieved by investigators using a web browser. Here we describe the database and access tools, provide an overview of the data therein and outline future plans. This resource has provided a unique opportunity for the distributed, collaborative study of a genus from which relatively little sequence data are available. Results may lead to insight into how better to control these pathogens. The homepage of PGI can be accessed at http:www.ncgr.org/pgi, with database access through the database access hyperlink.

Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences.

A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily.

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.

Discrimination of Rhizobium tropici and R. leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE).

With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize.

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum 'SES 208' (2n = 64, x = 8), thus permitting relations to homology groups ("chromosomes") of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

tRNA intergenic spacers reveal polymorphisms diagnostic for Xanthomonas albilineans.

A PCR-based detection system was developed for Xanthomonas albilineans, a pathogen of sugarcane, and other related xanthomonads, using the conserved sequence of two adjacent tRNA genes and the variable length and sequence of the spacer region between them. An appropriate region was identified as follows: tRNA genes with the same anticodon from a wide variety of bacteria were aligned and the most frequent base at each position was chosen to derive primers that would anneal to the gene in either orientation. Pairs of such primers were screened against various Xanthomonas species and members of related genera using PCR at low to moderate annealing stringency. A subset of these pairs of tRNA consensus primers gave one or more PCR products which generally displayed interspecific length variability. The primer pair 5'-3' tRNA(ala) and 3'-5' tRNA(ile) showed interspecific length polymorphisms between X. albilineans and all other Xanthomonas species examined. These PCR products were cloned and sequenced from four isolates of X. albilineans and four isolates from different pathovars of X. campestris, and the spacer length variation confirmed. Specific tRNA gene primers were derived from the tRNA gene sequences. These primers yielded a PCR product of a characteristic length within most Xanthomonas species and pathovars tested. When a primer that projected from tRNA(ala) into the 3' end of the variable intergenic spacer was used with a tRNA(ile)-specific primer, PCR was a very sensitive diagnostic test for X. albilineans-infected sugarcane and gave no product or only a faint product with other species of bacteria. The specificity of this PCR-based detection system was further enhanced by a nested PCR reaction that took advantage of the fact that the tRNA(ala)-tRNA(ile) region was found to be embedded in a 16S rRNA-23S rRNA gene spacer. By amplifying the region between the 16S rRNA gene and tRNA(ile) or between the tRNA(ala) and the 23S rRNA gene, the subsequent nested PCR product was shown to be X. albilineans-specific.

Genetic variability of Paecilomyces fumosoroseus isolates revealed by molecular markers.

Paecilomyces fumosoroseus (Deuteromycotina:Hyphomycetes) is a fungus that is potentially useful for the bio-control of economically important agricultural pests, such as whitefly (Bemisia tabaci). Arbitrarily primed PCR and PCR with tRNA consensus primers were used to analyze genetic variability among 27 P. fumosoroseus isolates, 15 of which came from the same host, B. tabaci, one P. lilacinus isolate, used as an outgroup, 9 previously unidentified Paecilomyces isolates. Fifteen 10-mer oligonucleotide primers of arbitrary sequence revealed 322 scorable binary characters. Principal coordinates and cluster analysis of characters showed that most of the P. fumosoroseus and Paecilomyces sp. isolates were in three phenetic groups with > 65% internal similarity. Two of the three arbitrary phenetic groups were closely related (76% similarity) with the third group quite different (only 14% similarity) from the first two. The phenetic groups did not correlate with geographical origin or host species. Genetic variability of isolates infecting whitefly in Florida was detected. Isolates from B. tabaci were represented in two of the three groups, and different genotypes were identified even when they were isolated from an epizootic population in India and Pakistan. There was no evidence of host-specific selection of genotypes, as has been shown in other entomopathogenic fungi. Three isolates morphologically classified as P. fumosoroseus were clustered in a phenetic group which displayed only 14% similarity to the other isolates of this species. Seven isolated that presented problems for morphological classification were found to be similar or, in three cases, identical to other P. fumosoroseus isolates that dit not present problems for morphological classification.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA markers for differentiating isolates of Paecilomyces lilacinus.

Paecilomyces lilacinus is an agent for the potential biological control of soil nematodes. Arbitrarily primed PCR was used to fingerprint the genomes of 28 isolates of this fungus. Most (72%) of the isolates originated from soil of different regions of Brazil. Fourteen 10-mer oligonucleotide primers of arbitrary sequence revealed 293 scorable binary characters. Distinct genotypes were obtained for each isolate. Cluster analysis showed a high level of variability among these genotypes. The similarity among pairwise comparisons of the isolates varied from 84.3% to 7.6%, with a mean of 63.5%. No clearly defined phenetic groups were identified by cluster or multivariate analyses. No correlation with geographical origin or host was detected. In addition, PCR with four pairs of consensus tRNA gene primers was performed on a subsample of 12 P. lilacinus isolates, three P. farinosus isolates, two P. fumosoroseus isolates, and one isolate of P. amoenoroseus. An inferred phylogeny based on 112 binary characters obtained by tRNA-PCR showed a monophyletic group which contained most of the P. lilacinus isolates. In contrast, three isolates of P. farinosus were not in a monophyletic group under the inferred phylogeny. These results suggest that tRNA fingerprinting could provide a valuable tool which could be used to develop the molecular taxonomy of Paecilomyces, as morphological characteristics of asexual structures cannot entirely resolve species.